In the field of blood analyses, it is known to measure the velocity of sedimentation of the corpuscular part of the blood (ESR) in order to evaluate the presence, for example, of inflammatory pathological states.
The machines and techniques used to measure ESR are known, for example as those described in the European patent application EP-A-1098188, published in the name of the present Applicant, which use optical emission means and detection means disposed on opposite sides with respect to a measuring volume.
A blood sample to be analyzed is injected into the measuring volume, and its flow is stopped suddenly, causing a characteristic kinetics of optical density of the corpusculate part present in the sample examined, which kinetics is known as syllectogram.
The optical density can be measured in units of absorbance or transmittance. Units of absorbance are determined using Lambert-Beer's law, where the value of absorbance is calculated by A=−log(I/I°)*L, where I° is the power of the light incident on the sample being measured, I is the power of the light exiting from the sample being measured and L is the length of the optical path or track, that is, the thickness of the sample.
The detection means is associated with processing means that measures said characteristic kinetics of optical density of the sample examined and calculates the ESR value or the velocity of aggregation of the red corpuscles using specific algorithms, characterized by parameters of the machine itself and its measuring characteristics.
In order to carry out the calibration of the machine, it is known to use, in parallel manner, a traditional reference method, scientifically recognized for measuring the ESR on the same blood sample, for example the Westergren method.
When the Westergren method is used as a reference, the ESR values measured are particularly sensitive to the variations in temperature of the environment where the tests are carried out. In fact, these values measured are considerably affected by the variation in temperature at which the test is carried out, as analytically described by the Stokes formula, with which the velocity of sedimentation is calculated starting from the knowledge of the rouleaux, the density of the suspension fluid, the viscosity of the liquid, etc.
It has also been proved experimentally that 3-5 Centigrade degrees of temperature variation between one test and the other on the same sample are sufficient to lose measuring accuracy to a figure of 30-50%.
For this reason, the National Committee for Clinical Laboratory Standards (NCCLS, H2-A4 Vol. 20 n° 27, page 1 “Scope ESR procedures cannot be calibrated”) considers that the procedure for measuring ESR cannot be calibrated because the procedures for determining ESR are susceptible to a variety of errors.
Given that the phenomenon of erythrocyte sedimentation and aggregation, described by the syllectogram, is limited to fresh blood and is transitory, as things stand at present, it is not possible to achieve materials for the standardized calibration of this test.
Even though Westergren remains the reference method for measuring sedimentation, it should also be noted that this method is extremely laborious, it is easy to make mistakes, it presupposes that the test tube containing the blood sample is perfectly vertical during the analysis, it can be performed at most within four hours after the blood sample has been taken, and it takes a much longer time for analysis compared with an automatic machine of this type.
Some producers have made and proposed controls to be used on different measuring systems for erythrocyte sedimentation: from the glass tube for Westergren to other instruments that measure ESR. With these controls, however, different ESR values are obtained for every measuring system in which they are used. Therefore, the measurements done with different systems on the same blood sample are different from each other according to the measuring system used, while the aim of a calibration should be to supply, for the same blood sample, an aligned ESR value, that is, repeatable and in conformity with ESR values measured in different environments, irrespective of the environment in which the measuring means is used.
From the published patent application EP-A2-0887637 it is also known to use spherical particles of synthetic polymers, having an average diameter comprised between about 1 and 8 micron, a restricted distribution of particles and a low refraction index, from about 1.35 to 1.45, in order to calibrate flow cytometers, in which the size, diameter and volume of red corpuscles, reticulocytes, white corpuscles and platelets contained in a blood sample are counted and measured.
This known calibration method is valid for flow cytometers or corpuscle-counters in which, typically, the cells or other biological particles having extremely small sizes, typically between 1 and 10 micron, flow in a liquid current, so that every particle, virtually one cell at a time, passes through a detection region where, on each occasion, the physical or chemical characteristics are measured, in this case the number, diameter and/or the volume.
Such flow cytometers are not able to evaluate the variation in optical density due to the sedimentation of particles present in a blood sample in a detection zone because they are able to analyze only the chemical-physical characteristics of one particle at a time, but not to detect a phenomenon like erythrocyte sedimentation, which involves a mass of particles.
Moreover, the use of this calibration technique with individual particles does not allow, in any way, to simulate and reconstruct the development of an optical density of a blood sample to be analyzed.
One purpose of the present invention is to perfect a method that allows the calibration, setting or alignment with respect to known values of machines for analyzing blood parameters connected to the density of the blood, such as the erythrocyte sedimentation rate and/or the index of the aggregation of the red corpuscles, known in literature as the M-index, in order to obtain measurements whose overall alignment comes within a restricted range of values, for example ±10%, in a univocal, repeatable and absolute manner, without depending on the temperature or other environmental factors, and which does not have the disadvantages of the state of the art.
The Applicant has devised, tested and embodied the present invention to overcome the shortcomings of the state of the art and to obtain these and other purposes and advantages.